Disclosed herein are polynucleotide sequences useful for producing transgenic plants with increased glycine-betaine content and methods of using such sequences for producing transgenic plants and seed. Such sequences are useful for producing transgenic plants with increased tolerance to stresses such as water-deficit and cold.
Stress, such as water-deficit, cold, heat, nutrient deficiency and the like, can have many adverse effects on plant performance such as yield reduction, increased susceptibility to disease and pests, reduced plant growth and reproductive failure. Considering the complexity of stress response in land plants, especially during conditions that produce water-deficit or cold, relatively few genes specifically associated with this aspect of physiology have been identified. It would be of benefit to the art to increase the number and variety of genes involved in regulating water use or temperature tolerance in plants, more particularly, in maize plants, and even more particularly in maize plants experiencing water-deficit and/or cold.
Glycine-betaine (N,N,N-trimethylglycine) is an osmoprotectant metabolite. Osmoprotectant metabolites, including betaines, such as glycine-betaine, sugars, sugar-alcohols, and amino acids, such as proline, are known to accumulate in plants under water-deficit and other stressful conditions such as cold conditions. Historically, applications of osmoprotectants to seeds and plants has been shown to have beneficial effects upon stress tolerance. Allard et al. (WO 99/01032) found that application of glycine-betaine to wheat plants increased the freezing tolerance of the plants by several degrees and Mottram (U.S. Pat. No. 5,952,267) disclose the foliar application of glycine-betaine to cotton plants under water-deficit which resulted in an increased number of cotton bolls.
The pathways for the synthesis of glycine-betaine are similar in higher plants and microorganisms. In both kingdoms, a two-step oxidation of choline occurs to produce glycine-betaine via an unstable glycine-betaine aldehyde intermediate. Choline is ubiquitous in higher plants. In spinach, the first step conversion of choline to glycine-betaine aldehyde utilizes a ferredoxin dependent choline monooxygenase. In E. coli, a membrane bound choline dehydrogenase performs this step. The second step, conversion of the unstable aldehyde to glycine-betaine, is carried out by glycine-betaine aldehyde dehydrogenase. This enzyme has been found to share strong similarity between plant and bacterial species.
Spinach, sugar beet and some varieties of maize are examples of higher plants in which glycine-betaine is found to accumulate under water-deficit stress. In contrast, many other plants, such as tomato, tobacco, rice and some varieties of maize, do not accumulate significant amounts of glycine-betaine, regardless of growing conditions.
Hanson et al., (U.S. Pat. No. 6,310,271) disclose tobacco transformed with a choline monooxygenase gene which exhibited increased accumulation of glycine-betaine. The transgenic plants also demonstrated increased tolerance to irrigation with saline solution when compared to non-transgenic controls. Bulow et al., (PCT Publication WO 98/26801) disclose the use of an E. coli choline dehydrogenase gene to impart increased freezing and choline tolerance in transformed potato plants. Allen et al., (U.S. Application No. 2002/0123118A1) disclose the proposed use of choline oxidase, L-allo-threonine aldolase, phosphoserine phosphatase and sarcosine oxidase genes for altering the levels of glycine metabolism in a transformed cell. Adams et al., (U.S. Pat. No. 6,281,411; incorporated herein by reference in its entirety) disclose naturally occurring metabolites, such as glycine-betaine (Wyn-Jones and Storey, 1982) that are osmotically active and/or provide some direct protective effect during drought and/or desiccation.
We have discovered DNA useful for the production of a transgenic plant with increased glycine-betaine. As used herein “GB1” is the name of a protein and its homologs, e.g., a protein at least 40% identical to GB1, the expression of which results in increased glycine-betaine in plants and “gb1” is the name of the DNA coding sequence and its homologs encoding and used to express the GB1 protein. “GB” is used herein to refer to the glycine-betaine metabolite.